Two-dimensional electrophoresis - MS is being used to identify the proteins from human mitochondria. To date 20 proteins have been measured and we plan to measure a large number of additional proteins. A high proportion of the proteins measured have been positively identified by comparison of the enzymatic fragmentation patterns with a fragmentation data base generated from the protein data base. In addition, we are seeking to elucidate posttranslational modifications (e.g., glycosylation, phosphorylation) with shifts in the positions of protein spots in 2-d gels. It is ultimately planned to compare the distribution of mitochondrial proteins from normal individuals with those from individuals suffering from metabolic diseases. An improved protein identification algorithm was devised and a computer program was written for this purpose. We are using the mitochondrial protein data to optimize our strategies for protein identification.